This is an NCRR Competitive Revision Application (NOT-OD-09-058 - 'Enabling RPGs to Leverage NCRR Center and Center-like Programs') submitted as a supplement to R01 AI39480 (Mechanism and function of the regulatory protein LAG-3). The goal of the parent R01 grant is to determine the role and molecular mechanism of LAG-3, a key negative regulatory T cell protein. Regulatory T cells (Tregs) are the primary mediators of peripheral tolerance and play a pivotal role in preventing autoimmune diseases and limiting chronic inflammatory diseases. There are two types of Foxp3+ CD4+ Tregs;'natural'Tregs (nTregs) and 'induced'Tregs (iTregs). LAG-3 is required for the maximal regulatory activity of both subpopulations. The Aims are outside the scope of the parent R01 but germane to its goals and overall focus on LAG-3 and Tregs. Supplementary Specific Aim A: Generation of Cre-reporter lines to facilitate the analysis of LAG-3 function in nTregs versus iTregs. The relative contribution of nTregs and iTregs in modulating immune reactions is not known, primarily because there are no tools or markers available that can reliably distinguish between these key regulatory subpopulations. Novel Cre-reporter lines will be generated that will allow for the identification, tracking and functional analysis of nTregs and iTregs, and thus the dissection of the role of LAG-3 in these subsets. The proposed strategy, which we refer to as FATCAT (Fulcrum gene Assisted Temporal Conditioning of Alleles Targeted), capitalizes on the temporal distinction in the initiation of Foxp3 expression by nTregs (in the thymus) and iTregs (in the periphery). These mice will be used in combination with a LAG-3 conditional knockout mutant to assess the role of LAG-3 in nTregs versus iTregs. Supplementary Specific Aim B: Defining the role of two novel molecules in Treg development and function. Two novel molecules have been found to be upregulated in Tregs compared with conventional CD4+ T cells (Tconv). This Aim will assess their contribution to Treg development and function, and determine if any link exist between their function and LAG-3. Targeted, floxed B6 ES cell clones will be obtained from the NCRR resource, KOMP. Mutant mice will be generated and crossed with Foxp3-Cre to restrict deletion to Tregs or use the Cre-reporter mice generated in the Aim above to restrict deletion to specific Treg subsets. The tools and experiments outlined above will greatly accelerate progress in the parent grant, and will provide new insight into the development and function of critical Treg subpopulations. Valuable tools and resources will be generated and made available to the scientific community through the NCRR-supported KOMP and MMRRC repositories. This revision also leverages ES cell reagents and expertise from the KOMP respiratory and its staff, respectively. Lastly, this revision achieves several ARRA objectives including the hiring and retention of personnel that will conduct the proposed studies, and contracting services from NCRR resources. PUBLIC HEALTH REVEVANCE: A greater understanding of the mechanism of LAG-3 function may lead to novel approaches for T cell specific therapeutic intervention. Furthermore, developing tools that allow us to directly assess the relative contribution of different Treg subsets in modulating a variety of disease situations is critical to develop more effective and appropriate therapies to improve human health.